IMMUNOLOGICAL DETECTION OF TISSUE TRANSGLUTAMINASE AUTOANTIBODIES IN COELIAC DISEASE

Abstract

120 Serum reticulin and endomysial autoantibodies are highly coeliac disease(CD)-specific. The autoantigens are preserved among both rodents and primates, including humans. A calcium-dependent enzyme tissue transglutaminase (tTG) has been shown to represent the predominant endomysial autoantigen in CD. We evaluated whether IgA-class tTG autoantibodies might be a useful tool in CD diagnosis. Sera of 134 patients with untreated CD and 207 disease controls were studied. tTG in phosphate-buffered saline or in calcium-containing Tris-buffered saline, pH 7.5, was used as antigen in an enzyme-linked immunosorbent assay (ELISA). Serum IgA-class endomysial antibodies (HUC-ab) were determined by an indirect immunofluorescence method using human umbilical cord as substrate and fluorescein-conjugated (FITC) antibodies as secondary antibody. IgA-class gliadin antibodies (AGA) were tested with an ELISA method. Calcium-treated and - untreated Western blots were performed with CD patient serum IgA, healthy control serum IgA and with mouse monoclonal tTG antibody CUB 7402 (MAb tTG). The antigen detected by MAb tTG was examined with the indirect immunofluorescence method using rhodamine-conjucated (TRITC) antiserum as secondary antibody. CD associated IgA did not specifically detect calcium-untreated tTG in ELISA. However, calcium-treated tTG autoantibody ELISA test proved to be highly sensitive, as 127 of 134 (95%) untreated CD patients were positive. The best cut-off limit was calculated to be 10 arbitrary units. Thirteen out of 207 disease control patients had slightly elevated titres (specificity 94%). Ten out of thirteen IgA-deficient CD patients were detected by IgG-class tTG autoantibody ELISA. The sensitivity for the IgA-class HUC-ab test was 92% and the specificity 100%. The results for AGA were 84% and 89%, respectively. tTG autoantibody ELISA test correlated better to the HUC-ab test than to the AGA test. MAb tTG detected proteins of higher molecular weight in immunoblot with calcium-treated tTG than in blot with untreated tTG. Double immunofluorescence staining (FITC/TRITC) with CD patient serum and with MAb tTG showed a complete overlapping in human umbilical cord, including both vessel smooth muscle endomysium and Wharton's jelly fibroblasts. In conclusion, tTG autoantibodies are highly sensitive and specific indicators for untreated CD. Calcium is needed for specific antibody detection. ELISA for the identified autoantigen allows an economical and rapid large-scale screening for CD. It also eliminates the observer bias always possible in immunofluorescent techniques.